Objective This experiment was conducted to investigate the effects of composite probiotic preparations on in vitro rumen fermentation gas production, gas composition, fermentation parameters, and nutrient degradation rate in dairy cows using the in vitro gas production technique, and to provide a reference for the scientific application of the composite probiotic preparations in the diet of dairy cows.

Method The experiment selected three multiparous lactating Holstein cows with permanent rumen fistulas as donor animals for rumen fluid collection. The composite probiotic preparation consisted of Pediococcus pentosaceus, Pediococcus acidilactici, Bacillus amyloliquefaciens, Pichia farinosa, and Dekkera bruxellensis, with a viable count ≥ 2×10⁸ cfu/g. A single-factor completely randomized design was adopted, and it was divided into 4 groups. The total mixed ration substrates were supplemented with 0 g/kg (CK group), 0.5 g/kg (W1 group), 1.0 g/kg (W2 group), and 1.5 g/kg (W3 group) of composite probiotic preparations. Each group had 6 replicates, and the in vitro culture was conducted for 48 hours.

Result 1) The W2 group exhibited the highest cumulative gas production, which was significantly greater than that of the CK group at 4, 24, 36, and 48 h (P<0.05), greater than that of the W1 group at 12 h (P<0.05), and greater than that of the W3 group during 12–48 h (P<0.05). With the increase in the addition amount of the composite probiotic preparations, the gas production volume increased quadratically from 12 hours to 48 hours (P<0.05). 2) Compared with the CK group, methane content in the gas was significantly lower in the W1, W2, and the W3 groups (P<0.05), and the oxygen and nitrogen contents were significantly higher than that of the CK group (P<0.05). 3) With the increase in the addition of composite probiotic preparations, the degradation rate of crude protein (CP) increased linearly (P<0.05), the degradation rate of starch decreased linearly (P<0.05), and the degradation rate of neutral detergent fiber (NDF) increased linearly and quadratically (P<0.05). The protein degradation rate in the W1 and W2 groups was significantly higher than that of the CK group (P<0.05), and the NDF degradation rate in W3 group was significantly lower than that of the W1 group and W2 group (P<0.05). 4) With the increase in the addition of composite probiotic preparations, the acetic acid content and total volatile fatty acid (TVFA) content in the artificial rumen fluid increased quadratically (P<0.05), while the isovaleric acid content decreased quadratically (P<0.05).

Conclusion Under the experimental conditions, adding the composite probiotic preparations to the diet can increase the gas production volume of the artificial rumen, reduce the methane production potential, and improve the degradation rates of NDF and CP, optimizing the rumen fermentation-related parameters; the appropriate addition level of the composite probiotic preparations is 1 g/kg of the diet.